Molecular Mechanism Of Stress Response Of Catalase In Arabidopsis

Plants often suffer from various stresses during their growth.Salt stress is one of the main abiotic stresses that plants often encounter.Salt stress causes excessive accumulation of reactive oxygen species(ROS)in cells,leads to oxidative stress,and affects plant growth and development.Hydrogen peroxide(H₂O₂)is one of the important reactive oxygen species,and its excessive accumulation will cause toxic effects on plant cells.Catalase(CAT)is one of the main enzymes that scavenge H₂O₂in the plant's oxidative defense system.There are three types of catalase proteins in Arabidopsis:CAT1,CAT2 and CAT3,which have high similarity and play important roles in plant response to oxidative stress.However,it is unclear whether different catalases have the same function under stress,what the regulatory mechanism is and the specific mechanism of different catalase response to stress is not clear.In this paper,specific monoclonal antibodies?-CAT2 and?-CAT3,polyclonal antibody?-Catalase,and plant materials of cat2,cat3,cat1/3,cat2/3,cat1/2/3 and many other mutants obtained from the lab's early work were used.Western blot,RT-PCR,yeast two-hybrid,and bi-molecular fluorescent complementarity(Bi FC)were used to test the response of different catalase enzymes to stress,and to explore the regulatory mechanism.Western blot detection results showed that under 150 m M Na Cl treatment and recovery,CAT2was induced during the stress treatment period in Arabidopsis thaliana wild-type materials,but CAT3 was induced extensively during the recovery period after salt stress treatment.The response of CAT1,which is analysed by using?-Catalase antibody in cat2/3 double mutant,was consistent with that of CAT2,but different from CAT3.Under the treatment of mannitol and methyl viologen(MV),which can simulate drought and oxidative stress,the responses of CAT2 and CAT3 were similar with that of salt stress.The analysis of RNA level showed that different responses of CAT2and CAT3 to salt stress may be regulated at transcription level.In order to further verify the mechanism of catalase response to stress,pro CAT2:CAT2-p CAMBIA3301,pro CAT2:CAT3-p CAMBIA3301,pro CAT3:CAT2-p CAMBIA3301,pro CAT3:CAT3-p CAMBIA3301 and fluorescent protein fusion expression vectors pro CAT2:EYFP-CAT2-p CAMBIA3301,pro CAT3:Ds Red-CAT3-p CAMBIA3301 were constructed.The transgenic lines were obtained by agrobacterium inflorescence dip method.The subsequent experiments will be performed to further investigate the regulation mechanism of catalase response to salt stress.In this paper,we also studied the degradation of CAT2 and CAT3 through ubiquitin proteasome pathway and cellular autophagy pathway to explore the relationship between stress response and catalase degradation.The cell autophagy pathway,in which ATG-related(Autophagy-related)ATG2 and ATG7 mutations cause a large accumulation of CAT2 and CAT3under normal physiological conditions,but have less effect on the response to salt stress of CAT2and CAT3.The results showed that the degradation of CAT3 in the response to salt stress may be through the ubiquitin proteasome pathway.Yeast two-hybrid showed that the CAT2^M and CAT3^Mwhich have partial catalase activity interacted with F-box protein EBF2(EIN3 Binding F-box Protein 2)in ubiquitin proteasome.Bi FC experiments failed to confirm the interaction between CAT and EBF.The innovations of this experiment is that we distinguished different catalases at the protein level.It is proved that different catalases have different expressions under stress,the expressions may be regulated by the transcription level under salt stress,this implies that different catalases play different functions under stress.The degradation of catalase was preliminarily investigated,CAT3 may be degraded by the ubiquitin proteasome pathway in the response to salt stress,and the autophagy pathway that ATG2 and ATG7 involved have little effect on the catalase response under salt stress.These results have innovative value for exploring mechanisms of different catalase response to stress and degradation in Arabidopsis.